Abstract: The immune system of mucosal tissues must effectively protect the host from pathogen invasion, while facilitating homeostatic interactions with a diverse colonizing microbiota. A clear understanding of the key molecules and mechanisms that achieve this delicate balance remains incomplete, leaving a gap in critical knowledge. This investigation, AI32438, has focused on D-defensins HD5 (DEFA5) and HD6 (DEFA6) as key effector molecules of innate immunity. These D-defensins are the most abundant antimicrobial peptides of human Paneth cells and compelling evidence shows that dysrupted Paneth cell function increases susceptibility to enteric pathogens, as well as to chronic inflammatory bowel disease. Published studies previously funded by this grant have established significant knowledge about the structure, microbicidal activities, and biological functions of HD5. In contrast, comparatively little is known about its Paneth cell partner, HD6 - an abundant D-defensin that is exceptionally well-conserved among primates. We discovered that HD6 has a novel mechanism of protective action. Unlike HD5 and other defensin peptides that protect the host through microbicidal activity, HD6 does not kill microbes but rather protects the intestinal mucosa by blocking microbial invasion. Our recently published data support a mechanism that involves trypsin- mediated processing of inactive stored proHD6 that generates a mature peptide capable of binding to surface proteins of microbes, followed by self-assembly of additional HD6 peptide molecules to form microbe-entangling nanofibrils and nanonets, which prevent penetration into host cells. The objectives for this proposal are to define the molecular details of this process, including pinpointing the target on microbial surface proteins that initiates initial binding of HD6 peptides, elucidating the structural features of HD6 that promote self-assembly, determining how HD6 impairs fagellar-driven microbial propulsion to mediate protection, and illuimate the consequences of HD6 binding to microbial antigens in shaping mucosal immune respones. We propose a combination of complementary approaches to accomplish these goals. By determining the fundamental features of HD6 function, these experiments will characterize a novel mechanism of protection and mucosal homeostasis afforded by the innate immune system. Successful completion of these studies will have broad impact on our mechanistic understanding of mucosal innate immunity will fill a void in knowledge on a conserved, highly abundant human defensin, as identification of the target for HD6 binding may reveal a new pathogen-associated molecular pattern (PAMP), and the structure-function analyses of HD6 will yield fundamental knowledge on mechanisms of peptide self-assembly.